Methods, Software, and Apparatus for Focusing an Optical System Using Computer Image Analysis

ABSTRACT

Methods, software, and apparatus for focusing an image in biological instrument are disclosed. Focusing elements are moved to various focus positions within a focus element travel range, and sample images are captured at the various focus positions. The sample images are resolved into subregions and an optimal focus position is determined based on the image intensity statistical dispersions within the identified subregions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/881,868 filed Jun. 30, 2004, now U.S. Pat. No. 7,394,943, which isincorporated herein by reference.

INTRODUCTION

The present teachings generally relate to methods, software, andapparatus useful in machine-vision-based focusing in imaginginstrumentation, for example in biological instruments.

During imaging operations such as biological analysis involvingnucleotide sequencing or microarray processing, photo-detectors are usedto detect signals arising from labeled samples or probe featuresresponsive to selected target analytes. These signals can take the formof electromagnetic emissions that are desirably analyzed to quantifysignal intensities arising from labeled samples or probe features andare subsequently resolved to quantitatively or qualitatively evaluatethe presence of a target analyte within a sample. Frequently, imagesassociated with such biological analyses are of a very high-resolutionto accommodate reading of very detailed images such as high-densitymicroarrays. High-density microarrays can have densities of 4 bindingsites or features per square millimeter or up to 10⁴ binding sites orfeatures per square millimeter. Binding sites can be positioned on thesubstrate by pin spotting, ink-jetting, photo-lithography, and othermethods known in the art of high-density deposition. Therefore, highlyprecise imaging is required. In order for image detectors to properlycarry out such precise imaging, it is desirable that the optical systemsassociated with the image detectors be in sufficiently sharp focus topermit the image detectors to differentiate between discrete portionswithin a particular target sample being imaged.

However, optical systems of desirable cost and complexity, suffer fromvarious types of optical aberrations that complicate the focusingprocess. A common optical aberration is field curvature aberration,which results in the situation where, for example, at one focalposition, the center of an image will be clear and in sharp focus, butthe edges of the image will be blurry and out of focus. Similarly underfield curvature aberration, at other focal positions, the edges can bemade sharp, while the center is blurred. This is because in commonoptical systems, the focal plane associated with an optical system isnot flat but rather can be substantially spherical in configuration.There are also higher-order aberrations in optical systems which may benon-symmetrical with regard to focus. In some optical systems, an imagedfeature size may increase faster on one side of sharp focus than on theother side.

Moreover, for various target samples that can be imaged in a biologicalinstrument, the surface of the sample to be imaged varies in height aswell. These varying heights within the sample result in differingdistances from the optical system's focal plane. Because sharpness offocus is related to proximity of the target to the focal plane, thesedifferences in height also create differences in focal sharpness atvarious points on the target sample.

Further, typical detectors used to image target samples are notone-dimensional point detectors but rather are two-dimensional andtherefore subject to tip and tilt. Ideally the planar surface of animage detector would be perfectly parallel to the surface of the targetsample. However, in real systems, there typically is some tip and/ortilt between the plane of the image detector and the surface of thetarget sample.

Frequently, imaging instruments employ imaging detection processes toimage target samples such as very high-density microarrays. In variousembodiments, microarray technology used in connection with the imaginginstrument is such that each element of the microarray emits lightproportional to the reactivity of the particular microarray element. Themicroarray is “read” by recording and analyzing images of the microarrayarray to quantify the light emitted by individual elements and therebyto identify analyte reactivity at each of the microarray elements.

In the case of a very high-density microarray, for example, lack ofsharpness or blur in an image increases cross-talk between individualmicroarray element signals, and reduces the instrument's ability todifferentiate among individual microarray elements. However, for aparticular biological instrument, it is typically a requirement that theinstrument be able to “read” (which is to say differentiate and measurethe intensity of) substantially every region in the target sample.Accordingly, adequate focus is typically required in substantially everyregion of the target sample's recorded image.

To overcome instrument focusing challenges such as sample-to-samplethickness variations, intra-sample surface height variations, fieldcurvature and other aberrations, and detector tip and/or tilt, it isnecessary to adjust the focus position of the optical elements in aninstrument to obtain a focal position of sufficient sharpness to permitthe instrument to operate properly. Moreover, for an imaging instrumentto operate efficiently, it is desirable that the instrument be able torapidly achieve focus. Therefore, there is a need for rapid, automaticfocusing in machine-vision devices that can compensate for opticalvariations, variations in the surface heights of target samples, anddetector tip and/or tilt.

SUMMARY

Images of a target sample are recorded while the target sample and/or anoptical system associated with the target sample are moved through aseries of positions bounding an ideal focus position. An analysis of therecorded images produces a map of the target sample's surface relativeto the optical system's focus plane. In various embodiments, the targetsample's surface height distribution is analyzed to select asubstantially optimal focus position for the target sample.Additionally, the present teachings can provide information regardingtarget sample physical defects, thereby providing beneficial samplequality control procedures in addition to automatically focusing opticsfor imaging a target sample.

In various embodiments, the present teachings can provide a method forfocusing an image in a biological instrument including moving a focusingelement to a plurality of focus positions within a focusing elementmovement range, capturing a sample image of a target sample at theplurality of focus positions, resolving the sample image into aplurality of subregions, calculating image intensity statisticaldispersion values within the plurality of subregions, identifyingsubregion focus positions for the plurality of subregions based on thecalculated image intensity statistical dispersion values, anddetermining an optimal focus position based on the identified subregionfocus positions, wherein the target sample is a high-density microarray.

In various embodiments, the present teachings can provide a method forfocusing an image in a biological instrument including moving a focusingelement to a plurality of focus positions within a focusing elementmovement range, capturing a sample image of a target sample at theplurality of focus positions, resolving the sample image into aplurality of subregions, calculating image intensity statisticaldispersion values within the plurality of subregions, identifyingsubregion focus positions for the plurality of subregions based on thecalculated image intensity statistical dispersion values, anddetermining an optimal focus position based on the identified subregionfocus positions, wherein the target sample is a high-density biologicalsample container.

In various embodiments, the present teachings can provide a method forfocusing an image in an imaging instrument including moving a focusingelement to a plurality of focus positions within a focusing elementmovement range, scattering electromagnetic radiation off a surface of atarget sample, capturing a sample image of the target sample at theplurality of focus positions based on the scattered electromagneticradiation, resolving the sample image into a plurality of subregions,calculating image intensity statistical dispersion values within theplurality of subregions, identifying subregion focus positions for theplurality of subregions based on the calculated image intensitystatistical dispersion values, and determining an optimal focus positionbased on the identified subregion focus positions, wherein the targetsample is a high-density microarray.

In various embodiments, the present teachings can provide software forfocusing an image in an imaging instrument including instructions tocause an electromechanical movement to move a focusing element to aplurality of focus positions within a focusing element movement range,instructions to cause a digital camera to capture a sample image of atarget sample at the plurality of focus positions, instructions to causean image processor to resolve the sample image into a plurality ofsubregions, and instructions to calculate image intensity statisticaldispersion values within the plurality of subregions and to identifysubregion focus positions for the plurality of subregions based on thecalculated image intensity statistical dispersion values and todetermine an optimal focus position based on the identified subregionfocus positions, wherein the target sample is a high-density microarrayor high-density biological sample container.

In various embodiments, the present teachings can provide an instrumentfor analyzing biological samples including an electromechanical movementcoupled to and operable to move a focusing element to a plurality offocus positions within a focusing element movement range, a digitalcamera coupled to the imaging instrument operable to capture a sampleimage of a target sample at the plurality of focus positions, an imageprocessor operable to resolve the sample image into a plurality ofsubregions, and a digital processor operable to execute instructions tocalculate image intensity statistical dispersion values within theplurality of subregions and to identify subregion focus positions forthe plurality of subregions based on the calculated image intensitystatistical dispersion values and to determine an optimal focus positionbased on the identified subregion focus positions, wherein the targetsample is a high-density microarray or high-density biological samplecontainer.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are only intended for the illustration of various embodiments. Thedrawings are not intended to limit the scope of the present teachings inany way.

FIG. 1 illustrates a schematic diagram of an exemplary imaginginstrument including an imaging detector, optics, and a focusing stage;

FIG. 2 illustrates a magnified view of a portion of an image produced byan exemplary microarray-reader instrument;

FIG. 3A illustrates a composite cross section of exemplary microarrays,illustrating sample-to-sample thickness variations;

FIG. 3B illustrates an exemplary graph of inter-sample surface heightvariations;

FIG. 3C illustrates an exemplary curved focal plane resulting from fieldcurvature aberration;

FIG. 4 illustrates an exemplary graph of inter-sample-surface heightvariations in the presence of image detector tip and/or tilt;

FIG. 5A illustrates an exemplary separation of a sample image intosubregions.

FIG. 5B illustrates a plot of image pixel intensity standard deviationversus focus position and corresponding exemplary images;

FIG. 6 illustrates plots of subregion image spread versus focus stageposition. Extreme foci are identified as curves showing maximum andminimum focus positions.

FIG. 7A illustrates a subregion focus curve pattern in which 100 percentof subregion foci can be located within the focus window;

FIG. 7B illustrates a subregion focus curve pattern in which only 80percent of the subregion foci can be located within a focus window.

FIG. 7C illustrates a subregion focus curve pattern in which only 20percent of subregion foci can be located within the focus window;

FIG. 7D illustrates a subregion focus curve pattern in which 85 percentof subregion foci can be located within the focus window and malformedsubregion focus curves suggest a problem with subregion image areas;

FIG. 8 illustrates an exemplary flow diagram for performing autofocusprocesses; and

FIG. 9 illustrates an alternative embodiment using electromagneticradiation to generate scattered light for producing an image of thetarget sample at an image detector.

DESCRIPTION OF VARIOUS EMBODIMENTS

Reference will now be made in detail to some embodiments, examples ofwhich are illustrated in the accompanying drawings. Wherever possible,the same reference numbers are used throughout the drawings to refer tothe same or like parts.

In various embodiments, the present teachings provide the ability toimage very high-density microarrays with sufficient resolution todifferentiate individual microarray elements. Instruments consistentwith the present teachings employ high-density image detectors, such ascharge coupled devices (CCD) and high resolution imaging optics designedto maximize the number of array elements that can be measured in aparticular image. In various embodiments, the optical system is designedto image the surface of a flat substrate on which the microarray hasbeen “printed” using high density spotting technology. In variousembodiments instruments image the microarrays using either or both offluorescent (FL) or chemiluminescent (CL) modes of the microarrays.

In various embodiments, reaction plates contain biological reagents suchas nucleic acid materials, primers, and probes, are imaged in connectionwith various biological assays to determine, for example a geneticsequence of a particular biological sample. It is understood that suchreaction plates can be standard 96, 384, 1536 or greater well trays.Moreover, biological reactions taking place in custom multi-well trayscan be imaged as well.

In various embodiments, capillary arrays such as those used inconnection with electrophoretic separation systems are imaged tofacilitate biological analyses performed in connection with the arraysof capillaries. In various embodiments microfluidic cards can be imagedas well. Similarly, arrays of tubes, in which biological analyses areperformed can be imaged consistent with the present teachings.

Specific aspects of the present teachings are described below in thecontext of exemplary microarray reader embodiments. However, it isunderstood that the present teachings are not limited in scope tomicroarray reader embodiments but can be used in connection with imagingof any type of biological analysis technique including, but not limitedto, those enumerated above. To clarify several terms used to disclosethe present teachings, several definitions are set forth below.

The term “optical system” as used herein refers, in various embodiments,to lenses or other optics used to manipulate and/or focuselectromagnetic radiation. However, the present teachings also apply tothe manipulation of particles such as electrons for example usingelectromagnetic fields to manipulate the paths of electron beams inelectron microscopy, in a manner analogous to lens refraction.Accordingly, as used herein “optical system” encompasses any system usedto aid in focusing of electromagnetic radiation or particle beam(s) forthe imaging of a target sample.

The term “target sample” as used herein refers to an object that is tobe imaged. In various embodiments, the target sample is a high-densitymicroarray, but it is understood that a target sample could be anyobject that is to be imaged by an imaging instrument consistent with thepresent teachings. Other target samples include other biological samplesand any object to be imaged using any type of microscopy, includinglight microscopy and electron microscopy.

The term “focal plane” as used herein refers to the set of points inspace at which distance the target sample is in substantially sharpfocus. In various embodiments, the focal plane associated with thepresently described optics is not two-dimensionally planar but ratherresembles a portion of a sphere in its configuration.

The terms “subregion focus position,” “subregion peak,” or “subregionmaximum” as used herein refer to the focus stage (or other opticalsystem movable element) position corresponding to the sharpest focus fora particular subregion. In various embodiments, a subregion focusposition is identified as the peak or maximum point on thesubregion-image-intensity-spread to stage-position curve. In variousembodiments, the subregion focus position is determined by recordingimages at a plurality of positions through the movement of the focusstage and calculating the standard deviation of pixel intensities withinthe subregion. In these various embodiments, the position at which thestandard deviation of pixel intensities within a subregion is highestcorresponds to the “subregion focus position.”

The terms “extreme foci” or “extreme subregion foci” as used hereinrefer to the nearest and most distant subregion focus positions to theimaging device, within a particular window of focus positions.

The term “focus window” as used herein refers to the window of distancesof the surface of the target sample from the imaging device in whichsubstantially all regions of the target sample can be imaged withsufficient sharpness or clarity. The absolute size of the focus windowcan also be referred to as “depth of field” or “depth of focus,” whichis the range of target sample surface distances from the focal planewithin which specific portions of the target sample can be imaged withan acceptable level of sharpness.

The term “statistical dispersion” as used herein refers to a measure ofnumerical diversity within a particular set of numbers. Statisticaldispersion is zero for a set of identical numbers and increases asdiversity among the set of numbers increases. Measurements ofstatistical dispersion include various examples. One measure ofstatistical dispersion is range, which is the difference between thehighest and lowest number values in the set of numbers. Anothermeasurement of statistical dispersion is “standard deviation,” which isthe square root of variance. Various algorithms are known for computingthe standard deviation and variance of a set of numbers.

The term “image intensity statistical dispersion” as used herein refersto a measure of numerical diversity of pixel intensity values for aparticular image or subregion within an image. Image intensity valuescan be obtained electrically from a pixel in a CCD or other detector orin connection with, for example, an electron beam detector in the caseof electron microscopy.

FIG. 1 illustrates a schematic diagram of an exemplary imaginginstrument 100 including an image detector assembly 110, optics 108, anda remotely controlled focus stage 102. In various embodiments, the focusstage 102 is a remotely controllable, motorized focus stage used toposition a target sample on sample platform 103 relative to the focalplane of the imaging system's optics 108. In various embodiments, thefocus stage 102 is positioned relative to the optics 108 and imagedetector such that predicted approximate focus positions are locatednear the focus stage's center of travel. In various embodiments, thefocus stage 102 is provided with sufficient range of travel to ensurethat the target sample can be moved from a location below the optimalfocus position to a location above the optimal focus position. Invarious embodiments, additional focus stage travel is provided toaccount for sample-to-sample focus variations and/or different focalpositions for different imaging modes. It is understood that positionsof the lenses or detector could be adjusted rather than adjusting theposition of the focus stage without departing from the teachings of thepresent invention.

As set forth below, in various embodiments, there is a relationshipbetween the focus position for electromagnetic energy of differentwavelengths. Therefore, in various embodiments, once the instrument isfocused in one wavelength, a calibrated offset can be used to positionthe instrument to a focused position in another other wavelength. Invarious embodiments, focusing can be performed without an additionalautofocus step by merely moving the focus stage 102 by the calibratedoffset distance by which the wavelengths differ.

In connection with the imaging system 100 of FIG. 1, in variousembodiments, autofocus operations are performed as follows. A microarraysample is placed atop the remotely controlled focus stage 102 on focusstage platform 103. The focus stage 102 is mounted relative to imagingoptics 108. In various embodiments, the focus stage can be positionedatop a sample insertion stage that moves the sample from a load positionto the imaging position. In various embodiments, to ensure proper focusin the instrument, autofocus processes consistent with the presentteachings are performed prior to actually imaging the sample forbiological analysis. As more fully set forth below, the autofocusprocesses involve recording and analyzing a series of images taken atdifferent positions of the focus stage 102. The autofocus processesinvolve analyzing the images taken at the different positions toidentify a position for the focus stage at which substantially all ofthe target image is sufficiently sharply focused to allow the instrumentto operate properly. After autofocusing is complete, quantitative imagesof the sample are measured and analyzed to determine individual elementsignal levels associated with the sample for biological analysis.

FIG. 2 illustrates a magnified view of a portion of an image produced byan exemplary microarray reader instrument. The image shows a regularpattern of bright points on a dark background. The light regions arecreated by light emitted from individual array elements. The darkregions correspond to the interstitial regions of the microarraysubstrate. Individual microarray elements are separated by a smallnumber of pixels on a CCD camera. The uniformly spaced bright regions202, 204 correspond to individual microarray elements. In variousembodiments, as set forth below, computer image analysis is performed todetermine a sufficiently sharp focus position to extract individualmicroarray elements from such an image.

FIGS. 3A-3C illustrate several of the factors affecting autofocusoperations of the present teachings. Autofocusing processes consistentwith the present teachings compensate for sample-to-sample thicknessvariations (FIG. 3A), inter-sample height variations (FIG. 3B), andfield curvature effects (FIG. 3C). Due to field curvature of imagingoptics, a flat microarray surface presents challenges in obtaining animage of the microarray in which each region of the image is perfectlysharp. Field curvature causes the optimal focus at a particular point tovary as a function of the distance from the optical axis (assuming aflat microarray substrate). If the focus is optimized on the center ofthe image, the edges of the image will have poorer focus. Inter-sampleheight variations present challenges in simultaneously optimizing focuson a sample's high and low points. Inter-sample height variations tendto be random in nature indicating a need to characterize the high andlow points prior to imaging. Tip and/or tilt of the detector, and anon-flat sample substrate present challenges to having a substantiallyparallel sample and detector.

In various embodiments, an optical system is selected such that thedepth of focus is sufficiently large to accommodate field curvature andinter-sample height variations of the majority of typically encounteredsamples. After autofocus operations are performed, only portions of thetarget sample's surface are imaged at substantially sharp focus, butother regions are sufficiently close to focus to provide adequateimaging resolution. In various other embodiments substantially none ofthe points within the target sample are in substantially sharp focus,but nevertheless substantially all points within the target sample aresufficiently sharply focused to allow the imaging instrument to make animage of adequate quality. In various embodiments, the present teachingsprovide an autofocus algorithm that is capable of positioning the targetsample such that no region of the target sample falls outside theoptical system's depth of focus.

FIG. 3A illustrates a composite cross section of two exemplarymicroarrays, illustrating sample-to-sample thickness variations. It canbe observed that sample 306 has a thickness that is greater than sample308. Accordingly, an optimal focus distance for the two thicknesseswould be offset by approximately the thickness variation 302 shown inFIG. 3A. Additionally, thickness variations in the adhesive 310 or inthe thickness of the glass substrate 312 could result insample-to-sample thickness variations that can be compensated for on asample-by-sample basis by employing autofocus techniques consistent withthe present teachings. In various embodiments, the sample-to-samplethickness variation 302 is in the range of approximately Opm to 400 μm.

FIG. 3B illustrates an exemplary graph of inter-sample surface heightvariations. The mean plane 323 can be calculated by averaging the valueof the points along the inter-sample surface height variation curve 322.In this example, it can be observed that there are maximum points in thesample that are 120 μm above the mean plane 323 and minimum points thatare 80 μm below the mean plane 323. Accordingly in order for the maximumand minimum points all to be sufficiently sharp, an optical system forimaging the sample would require a depth of focus of approximately120+80 or 200 μm as shown in FIG. 3B.

FIG. 3C illustrates an exemplary curved focal plane resulting from fieldcurvature aberration. An optical axis 324 is shown that typically passesthrough the center of lenses associated with the imaging instrument'soptical system. Flat sample 328 is shown below focus plane 326. Toadequately focus the surface of the flat sample 328, the flat sample 328can be positioned upwardly into the focus plane 326.

FIG. 4 illustrates an exemplary graph of inter-sample-surface heightvariations in the presence of image detector tip and/or tilt. In variousembodiments, due to tip and/or tilt, a focus window 410 is parallel tothe plane of the image detector, but tipped and/or tilted with respectto the target sample plane 420. In various embodiments, minor tip and/ortilt deviations can be compensated for in connection with autofocusoperations consistent with the present teachings. It can be seen bycomparing FIGS. 3B and 4 that given similar inter-sample surface heightvariation curves, the introduction of tip and/or tilt can require agreater depth of focus, for example if tilt causes the higher points ofthe surface to become closer to the image detector or if the lowerpoints to become even further away.

FIG. 5A illustrates a sample calibration image 502 divided into 25subregions. In various embodiments, the image detector is a CMOS orCCD-type image detector that provides a matrix of pixel intensity valuescorresponding to an intensity of electromagnetic radiation received at apixel in the image detector. An indication of degree of sharpness in aparticular subregion can be described by the degree of diversity ofintensity values received at the pixels, or the pixel intensitystatistical dispersion. In various embodiments, a quantitative measureof pixel intensity variation is the standard deviation of pixelintensity in a particular subregion of the target sample image.Accordingly, consistent with the present teachings, during autofocusoperations, at various positions of the focus stage, target sampleimages are recorded and analyzed. To analyze the recorded images forgenerating the subregion foci, the recorded images are first dividedinto subregions 504.

In various embodiments, an image of dimensions approximately 2 k×2 kpixels in size image is divided into 25 subregions as shown in FIG. 5A.Binning is a process by which intensity values from two adjacent pixelsin a CCD are combined to reduce the amount of data associated with a CCDimage. In various embodiments 2×2 CCD camera binning is employed toreduce the amount of data necessary to be transferred from the CCD toexpedite autofocus operations. It is understood that 4×4 (or higherlevel) binning could be employed without departing from the scope of thepresent teachings.

It is understood that larger pixel arrays can be employed withoutdeparting from the present teachings. Moreover, it is understood thatany number of subregions can be employed without departing from thepresent teachings. In various embodiments, a 5×5 grid, yielding 25subregions can be selected for speed-optimization purposes and becauseit provides a sufficiently large number of subregions to yield optimalfocus positions, for example, even when there are bubbles in one part ofthe fluid of a target sample to be imaged and/or even if the imagedetector has some degree of tip and/or tilt with respect to the targetsample. In various embodiments, four, nine, sixteen, or thirty-sixsubregions can be employed. It is also understood that subregions neednot be symmetrical or of equal size. In various embodiments, subregionsizes are selected such that at least one illuminated microarray spot,for example a fiducial, is guaranteed to be in each subregion to providecontrast for performing image intensity statistical dispersioncalculations. In various embodiments, subregions are sufficiently smallthat some subregions may not contain any fiducials or illuminatedmicroarray spots. In these various embodiments, subregions notcontaining any contrasting pixels are discarded and not used forautofocus operations.

FIG. 5B illustrates a plot of image pixel intensity standard deviationversus focus position for an exemplary subregion of a sample imagerecorded for autofocus operations. It can be observed that when theimage standard deviation is greatest, i.e. approximately 113, thesubregion image is at its most sharply focused position. In this examplesubregion images 512 and 516, there is considerable blur caused byfactors including field curvature aberration, tip and/or tilt, and/ordifferences in sample thickness. By contrast, subregion image 514,corresponding to the peak pixel intensity standard deviation is insubstantially sharp focus.

FIG. 6 illustrates plots of subregion image variance versus focus stageposition. Extreme foci are identified as curves showing maximum andminimum focus positions. In various embodiments, the autofocus algorithmdetermines the proper focus stage position by using the two extreme focipositions. If the distance between the extreme foci is less than thefocus window, each region of the image can be adequately focused duringimaging. In FIG. 6, subregion curve 602 represents the low extreme focusstage position, while subregion curve 604 represents the high extremefocus stage position. In various embodiments, the optimal focus positionis chosen to be halfway in between the extreme foci, the focal positionfor the particular target sample being the average value of the extremefoci. In FIG. 6, the optimal focus position is focus stage position 603.

FIG. 7A illustrates a subregion focus curve pattern in which 100 percentof subregion foci can be located within the focus window. As in FIG. 6,the focus stage can be positioned at the average of the maxima of thesubregion focus curves that are the extreme foci, namely the midpoint ofthe maxima of curves 702 and 704.

FIG. 7B illustrates a subregion focus curve pattern in which only 80percent of the subregion foci can be located within a focus window. Herethe position of the focus stage is selected to be the midpoint of theextreme foci that fit within the focus window, maximizing the number ofsubregion peaks within the focus window. It is understood that themaximum number of subregion peaks in the focus window can be ascertainedin any way including calculating the number of subregion peaks in theparticular focus window as the focus window moves through the autofocusprocess and iterating through a set of possible focus window positionsand counting the number of subregion peaks within each possible focuswindow in the set.

FIG. 7C illustrates a subregion focus curve pattern in which only 20percent of subregion foci can be located within the focus window. Inthis case, the extreme foci are again selected such that the greatestpossible number of subregion curve maxima are positioned within thefocus window, nevertheless, the variation in position along the focusstage position axis indicates that the target sample cannot beaccurately imaged. In various embodiments, an imaging instrument wouldgenerate an error indication, indicating that only portions of thetarget sample could be clearly imaged. In various embodiments, multipleimages are made over a range of focal positions to separately image thesubregions in sharp focus, and thereafter a composite image of thesharply imaged subregions is composed.

FIG. 7D illustrates a subregion focus curve pattern in which 85 percentof subregion foci can be located within the focus window and malformedsubregion focus curves suggest a problem with subregion image areas. Itcan be observed that outlier subregion curves 710, 712, and 714 indicateabnormalities in the subregions corresponding to curves 710, 712, and714. In various embodiments, such abnormalities can indicate defects inthe target sample such as bubbles in a sample liquid associated with amicroarray. In various embodiments, the detection of such abnormalitieswill result in an error indication by the imaging instrument. In variousembodiments, the outlier curves are ignored. The outlier curves cancorrespond to defects in the target sample that do not impede properimaging of the target sample if the focus position corresponding to thenon-defective subregions of the target sample is used.

In various embodiments, a microarray sample or other target samplecomprises a relatively flat substrate submerged beneath a conditioningliquid. In various embodiments, the conditioning liquid is containedabove the substrate without any top cover. In various embodiments, theconditioning liquid is positioned between the substrate and atransparent cover glass surface sealed at the edges of the substrate.Various defects can arise during sample preparation that interfere withinstrument performance. Defects can include the presence of bubbles inthe conditioning liquids contamination of the cover glass surface orsubstrate, and various other irregularities that can affect imagequality. The present teachings provide a means for detecting thesedefects prior to measurement thereby providing an opportunity for theuser to correct the problem, or to flag the results associated withdefective regions of the array.

FIG. 8 illustrates an exemplary flow diagram for performing autofocusprocesses consistent with the present teachings. First the stage ismoved to a starting position below a predicted position of focus (step802). A predicted focus position can be obtained in any way, such as,for example an average focus position obtained by averaging a group offocus positions for a group of target samples of the type beingautofocused. It is understood that the stage could also be positioned inother alternative positions, such as above a predicted focus. Nextoptics are configured for autofocus mode (step 804). In variousembodiments, this involves positioning the filter between the stage andthe optics so as to filter out photons being reflected from the slidethat originate from the fluorescent excitation source. Next, an image ofthe target sample is recorded (step 806). Then the image is analyzed todetermine subregion image statistical dispersion data (step 808). As setforth above, in various embodiments the subregion statistical dispersiondata is obtained by dividing the image up into subregions andcalculating the standard deviation of the intensity of pixels within thesubregions. It is understood that any calculation representing imageintensity statistical dispersion can be employed without departing fromthe scope of the present teachings. In various embodiments, analysis ofthe statistical dispersion versus position curves begins once two ormore points have been acquired. With two points measured, the curves'slopes can be calculated permitting a prediction of whether a curve'speak lies above or below the focus positions that have been imaged(searched region). With three or more focal position points measured,each statistical dispersion curve is identified as either peaked orlying above or below the searched region. In various embodiments, ateach focus position in the autofocus operation, the previously detectedpeak positions are considered in connection with the known size of thefocus window of the optical system to determine an optimum placement ofthe focus window for the presently detected set of peaks.

Next, the peaks are counted that are observed in the variance toposition curves calculated at various positions (step 810). Next it isdetermined whether all peaks are located, whether the focus travel limitis reached, whether continued searching for peaks is warranted, orwhether the search region start position needs to be modified byreturning to step 802 and continuing to search from a position furtherbeneath focus. An error condition is identified when it is observed thatthe range of detected peaks exceeds one focus window. In thiscircumstance, further searching for peaks may or may not be warranteddepending on operator preferences (step 812). User preferences caninclude a permitted number of percentage of peaks that are permitted tolie outside of the focus window. In various embodiments, this wouldallow a microarray to be imaged notwithstanding a bubble in the targetsample or a dust particle on the surface of the target sample.Accordingly, it is understood that a focus result wherein fewer than100% of the peaks are located within the focus window can be employedwithout departing from the scope of the present teachings. If none ofthese conditions are true, then in various embodiments, the predictedfocus position is lowered (step 813) and the process continues at step802. If the step 812 conditions are observed, then in variousembodiments the high and low points for peaks within the focus windoware identified (step 814). Further, once the high and low points forpeaks within the focus window are identified in step 814 above, invarious embodiments, the high and low points are averaged and the stageis moved to the average position as the optimal focus position (step816). Other algorithms for determining optimum focus position based onthe distribution of peaks within the focus window, for example,selecting the optimum focus position by averaging the peak positions canbe employed without departing from the scope of the present teachings.

FIG. 9 illustrates an alternative embodiment using electromagneticradiation to generate scattered light for producing an image of thetarget sample at an image detector. An illumination source 105 isdirected at the sample such that only non-specularly reflected(scattered) light is imaged by the detector 110. In this case, noemission filter is used so the detector 110 observes light scatteredfrom the surface of the target sample on the focus stage platform 103.In the various embodiments illustrated in connection with FIG. 9,scattered light 906 is used to image a surface texture of the targetsample. This surface texture imaging can advantageously be used, forexample, in quality control operations on microarrays, whereby a nylonsurface of the microarray is imaged to determine whether the surfaceheight variations of the nylon fit within acceptable dimensions. It isunderstood that the various embodiments disclosed in connection withFIG. 9 can also be used, for example, in connection with lightmicroscopy and/or photography generally.

In various embodiments, the present teachings can provide imaging forhigh-density biological sample container that can present similarchallenges as high-density microarrays including sample-to-sample depthvariations, inter-sample height variations, and field curvature effects.The container can include any of known containers in the biologicalfield including reaction plates with high-densities of reaction wellssuch as 96, 384, 1536, 6144, etc. wells, custom multi-well reactionplates that are not standard consumables, i.e. SBS, a plurality ofcapillaries in an capillary array, such as a 96-capillary array, aplurality of individual sample tubes arranged in an array configurationsuch as tube strips, a plurality of individual sample locations in amicrofluidic card, such as 96 or 384 chambers that can be vacuum or spinloaded.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities, percentages orproportions, and other numerical values used in the specification andclaims, are to be understood as being modified in all instances by theterm “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that may vary depending upon thedesired properties sought to be obtained by the present invention. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a range of “less than 10” includes any and allsubranges between (and including) the minimum value of zero and themaximum value of 10, that is, any and all subranges having a minimumvalue of equal to or greater than zero and a maximum value of equal toor less than 10, e.g., 1 to 5.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “a microarray” includes two or more microarray.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

1. A method for focusing an image in a biological instrument, the methodcomprising: moving a focusing element to a plurality of focus positionswithin a focusing element movement range; capturing a sample image of atarget sample at the plurality of focus positions; resolving the sampleimage into a plurality of subregions; calculating image intensitystatistical dispersion values within the plurality of subregions;identifying subregion focus positions for the plurality of subregionsbased on the calculated image intensity statistical dispersion values;and determining an optimal focus position based on the identifiedsubregion focus positions, wherein the target sample is a high-densitymicroarray.